In many methods of biological analysis, a solid phase has to be separated from a liquid phase and subsequently washed. To wash the solid phase, a defined amount of buffer solution is pipetted into the reaction vessel containing the solid phase to suspend the solid phase in the buffer solution. The solid and the liquid phases are then separated. The liquid phase is then removed by suction (aspiration) and a new washing process begins. Usually a number of washing cycles are carried out, each including a suspension, separation and aspiration process.
The use of magnetic particles as a solid phase and separation by permanent magnets is known in principle. Permanent magnets attract the particles to the wall of the reaction vessel and hold them there.
Magnetic particles are often used in separation processes. There are many biological assay methods and purification methods in which magnetic particles are used. For example, immunoassay methods, nucleic acid hybridisation assays and the like. Magnetic particles can also be used in purification methods, to isolate particular components, proteins, nucleic acids, from the material in which they were contained. The particles can be used to separate certain components from a mixture, for example, because they are coated with a reagent with a specific affinity for the component. Magnetic particles can be drawn to, for example, the wall of a container in which the fluid with the magnetic particles was contained and the fluid can be removed and, optionally, be replaced with another fluid. Thus, the particles can be mixed with the fluid from which the specific component is to be removed, the component will bind to the magnetic particle, and a magnet can be used to separate the particles with the component from the remainder of the mixture in the fluid. Optionally the magnetic particles can be washed, and can be separated in another fluid. Or the component can be removed from the particles again into another fluid.
European patent application EP-A-0.136.126 describes a device for separation during solid-phase immunoassays. The bottom end of a reaction vessel containing magnetic particles is disposed between two permanent magnets. The axes of magnetization are at right angles to the wall of the reaction vessel, thus reducing stray magnetic fields:
International application WO-A-92/05443 describes a device for separating magnetic particles. The reaction vessels containing the magnetic particles are disposed in rows. Between the rows is positioned a magnetic block. The reaction vessels are disposed in the magnetic block such that two magnets-are diametrically opposite relative to the reaction vessel. The magnets have alternating polarities and their magnetization axes extend parallel. Separated particles are on only one side of the reaction vessel.
The U.S. Pat. No. 4,895,650, the contents of which is herein incorporated by reference, describes a separating device in which particles are separated by a permanent magnet. The magnet is on only one side of the reaction vessel. The relation between the level of test solution in the test-tube and the position of the magnet is focused on. The position of the magnet, more particularly its height, must coincide with the level of test solution in the reaction vessel, and is brought to the desired height by packing material in the bottom part of the device holding the magnet.
During an immunoassay, the fluid level in the reaction vessels after adding the required reagents is not necessarily uniform. For example, the level in the reaction vessel after adding conjugate solution may be lower than the level after adding washing buffer solution. The method of analysis described in this former U.S. Pat. No. 4,895,650 does not take these differences in level into account.
Known devices for separating magnetic particles have the disadvantage of requiring a relatively long time before all magnetic particles are separated from the liquid phase. Separation time may be considerable, particularly for larger volumes.
A device for rapid separation of magnetic particles is described in European patent application EP-A-0.317.286. In this device, the reaction vessel is surrounded by four permanent magnets (magnets 1, 2, 3 and 4), which are uniformly distributed around a reaction vessel. The direction of the magnetic field of magnets 1 and 3 is rotated through 180° relative to the direction of the magnetic field of magnets 2 and 4. This device has the disadvantage of requiring a relatively large number of permanent magnets to speed up separation. It also excludes many possible cell movements.
In the EP-B-0.644.425 patent is presented an analyser having a device for separating magnetic particles from a suspension, the separation device containing two permanent magnets between which the reaction vessel containing a suspension is located. For faster and more complete separation of the magnetic microparticles, the magnets are located diametrically opposite with respect to the reaction vessel and the pole axes of the magnets form an acute angle with the longitudinal axis of the reaction vessel. An aim of this invention is to provide an analytical device comprising a device for separating magnetic particles such that the magnetic particles in suspension can be rapidly separated even when the reaction vessel is filled to different levels. Another aim is to provide an analytical device for separating magnetic particles such that the magnetic particles in suspension can be separated in a focused manner.
If these documents can be considered for the separation of magnetic particles from a liquid of interest, they cannot authorize a performant mixing that could efficiently wash the particles and give them all chance to bind onto the magnetic particle's surfaces. This efficient step of mixing is absolutely necessary for purifying nucleic acid targets from a biological sample.
State of the Art Concerning the Mixing Step:
Purification methods for nucleic acid using magnetic particles have for example been described in various applications such as EP-A-0.757.106 and WO-A-96/41811. In these applications methods are described wherein a sample solution containing nucleic acids is treated with a chaotropic substance to release the nucleic acid. After releasing the nucleic acids from the biological entity in the lysis buffer, the nucleic acids are bound to the magnetic particles. Both particles coated with a target-specific probe as well as particles having a metal oxide coating (e.g. silica), giving a generic binding of all nucleic acids contained in the sample are used for this purpose. After binding the target, interfering components such as cell debris, enzymes, proteins anti-coagulants and salt are removed by washing the magnetic particles in a (set of) wash buffer(s). Finally, the purified nucleic acids are released from the particles by mixing the particles in a small volume of elution buffer.
For efficient washing and elution the magnetic particles need to be well dispersed and mixed in the relevant buffers. In general, this washing and elution process may be hampered by the aggregation or clogging of the magnetic particles either caused by the adsorption of specific components in the lysed sample (e.g. genomic DNA) or by residual magnetic dipole fields induced in the particles. In particular, the use of silica coated (magnetic) particles with samples that contain significant amounts of genomic DNA (whole blood, sputum, tissue), results in a tight pellet that is difficult to process.
Well-known methods for mixing (magnetic) beads in a liquid buffer are vortexing, sonification or pipetting. These methods however are difficult to automate, and/or give risk of sample to sample contamination by aerosol generation or they may degrade the nucleic acid target. Furthermore, these methods are not well suited for very small volumes of liquid (typically 0.01 ml) as may be required for the elution process.
The method and device according to the invention are especially suitable for use with isolation procedures, where, usually an ingredient is to be isolated in rather pure form from a relatively large volume of sample fluid, and concentrated into a smaller volume of another fluid to be suitable for further use.
In the case of a method for the isolation of nucleic acid such further use may be a nucleic acid amplification method or an assay for the detection of nucleic acid or both.
A method and apparatus for separating and resuspending superparamagnetic particles is disclosed in the application WO-A-91/09308. In this application it was disclosed that superparamagnetic particles may be aggregated and resuspended by subsequent application of different magnetic fields. First and second applications of the magnetic field could be provided with the same magnet, which was then rotated around the container containing the particles to a different location. Two spaced opposed electromagnets, however, could also be used. These electromagnets were energized alternately to produce the first and second magnetic fields that keep the particles in suspension and mix them with the fluid in which they were contained.
A method for the separation of magnetic particles from a fluid is disclosed in U.S. Pat. No. 3,985,649. The particles may be separated from a fluid by bringing the particles into close proximity with a magnet and moved through the liquid along the wall of a container. They may even be moved out of the liquid in this way and can be transported to a second container.
In U.S. Pat. No. 4,988,618, a device is described for use with assays wherein multiple small volume samples are tested at the same time. This type of assay can be performed in, for example, microtiter plates. Magnetic microparticles are present in each well of the microtiter plate. The device thus has multiple orifices and the orifices are each surrounded by multiple permanent magnets, preferably four. The resulting structure of magnets and orifices is rigid; the magnets are not intended to be moved and are mounted in a fixed relation with respect to themselves and to the base of the device. All magnetic are aligned and the field orientation of the magnets may be such that all magnets have the same field direction or neighbouring magnets have opposite field directions. The magnets orientation thus results in four spot attraction sites per orifice. The magnets are purely meant for separation purposes. It is disclosed in the patent that the device may further comprise means or agitating the reagents within the containers.
The applicant has already filed an international application WO-A-0 1/05510 which proposes a solution to improve the mixing. It relates to a method and device, which allows efficient mixing of magnetic or magnetizable particles in a fluid and optionally separation of the particles from said fluid. Use is made of magnetic field of opposite and changing directions. It has been found that, when magnetic or magnetizable particles in a fluid are subjected to these magnetic fields, the particles are, under the influence of the filed, efficiently contacted with the fluid. Such particles normally may tend to form a clot, which can prevent efficient mixing with a fluid. It has been found that, by subjecting the container in which the fluid and the particles are comprised, to magnetic fields of different and changing directions, the particles are efficiently separated from each other and drawn trough the fluid in such a way that a very efficient mixing process occurs. The method allows efficient mixing of particles with even very small fluid volumes. The method of the invention therefore has the advantage that it may save in, for example, washing fluids and may allow the reduction of the volume of fluid needed. Thus, for example in isolation procedures, the method of the invention allows the purification of reagents in high concentrations. Beside, whereas prior art methods can be laborious and time consuming; the method is fast and easy to perform.
Thus, provided with this application is a method of mixing, in one or more container(s), magnetic or (super) paramagnetic particles with a fluid, using more than one magnets, whereby the containers are subjected to magnetic fields with different and changing directions by moving the magnets with respect to the position of the container(s) and/or by moving the containers with respect to the positions of the magnets.
State of the Art Concerning the Concentration Step:
Many diagnostic tests are carried out after steps of extracting the target analytes from biological samples, of purifying in order to remove parasitic products which penalize the performance of the test, of concentrating the target analytes in order to increase the amount of analyte per unit of buffer volume, and of dissolving the target analytes in a buffer in order to make them chemically accessible.
In addition, in order to increase the sensitivity and the specificity of a test for demonstrating an analyte, it is sometimes necessary to reduce the volume of the buffer in which the copies of the analyte being sought are found, while at the same time conserving said analyte in its entirety.
Biologists have entirely conventional means for concentrating an analyte, in particular using centrifugation, filtration and/or magnetic sedimentation techniques. These techniques require transfers of solutions and manipulations of the analyte, which lead to an inevitable decrease in the amount of analyte that can be analysed.
For example, in centrifugation and magnetic sedimentation methods, the actual centrifugation or magnetic sedimentation steps may have to be repeated several times, the limit of the number of repetitions being set by the minimum volume of solution which can be easily and reliably handled with a conventional pipette. This minimum volume is of the order of about 10 micro litres. Below this, transporting it in “large” containers such as pipettes, flasks, etc loses liquid, and therefore analyte. In addition, there are problems of evaporation and of adsorption to the walls of the containers during these manipulations.
In the case of a low concentration of analyte in the starting sample, this may cause the complete disappearance of the analyte or a decrease in the amount thereof such that it may become undetectable.
Besides the abovementioned drawbacks, these manipulations are expensive in terms of material and take a lot of time. This remains a constant problem for many industrial applications, for example the detection of pathogenic micro organisms in a biological specimen or an industrial sample.
The international application, WO-A-02/43865, concerns a method for transporting an analyte present in a sample, a method for concentrating an analyte present in a sample, and a device for implementing said methods. The method for transporting an analyte present in a sample consists in preparing a solution from the sample wherein the analyte is fixed on magnetic particles; introducing this solution in a first container connected via a bottle-neck to a second container; displacing with a magnetic system the analyte fixed on magnetic particles from the first container to the second container via the bottle-neck; the second container being filled with all or part of said solution and/or with another solution.
Here again, the solution is provided to how to concentrate magnetic particles into a small volume of liquid. However the separation is not efficiently treated, especially if the magnet is of a smaller size compared to the first container, and the mixing is even not discussed.
A real need therefore exists for a method and a device for treating analytes bound to magnetic particles while at the same time conserving the amount of analyte present at the start, for example in order to increase the sensitivity and the specificity of diagnostic tests and of any chemical reaction directed towards the analyte, and to overcome the abovementioned drawbacks.
The present invention satisfies this need, and has not only the advantage of overcoming the abovementioned drawbacks, but also many other advantages, which those skilled in the art will not fail to note.
Thus none of the above-described documents presents a process offering the advantages of separating, mixing and concentrating molecules of interest from a liquid sample in only one container. Likewise, they do not propose a container having technical features authorizing the running of such a process. Regardless of the claimed method, it may be either manual or automatic, or operated by an automated device or not.
This is the main goal of the present invention to propose a process for manipulating magnetic particles that are suspended in a fluid, possibly containing a biological entity of interest, the magnetic particles being able to bind the entity of interest, the fluid being contained in a reaction vessel constituted by a large upper compartment with a funnel shape, an elongate lower compartment with a substantially constant cross-section and a closed base, consisting of:                a) subjecting the magnetic particles to two magnetic fields applied simultaneously to separate all said magnetic particles present in at least the upper compartment of the vessel from the fluid,        b) transferring the separated magnetic particles from the upper compartment to the elongated lower compartment,        c) removing the fluid from the vessel,        d) adding a washing liquid to the lower compartment,        e) subjecting the rest of the fluid to at least two magnetic fields applied successively with different and changing directions to wash all the magnetic particles present in the lower compartment, and        f) concentrating said magnetic particles in said lower compartment.        
In a particular configuration of the invention, the two magnetic fields applied simultaneously are generated by at least two magnets, the pole axis of the magnets forming together an angle different from 180°, preferably included between 30 and 150° and more preferably included between 60 and 120°.
In another particular configuration of the invention, the at least two magnetic fields applied successively are generated by at least two magnets, the pole axis of the magnets being parallel one to the other.
In a specific embodiment of the latter particular configuration presented just above, the successive magnetic fields with different and changing directions are applied to the vessel by moving the magnets with respect to the position of the vessel and/or by moving the vessel with respect to the position of the magnets.
Always in a particular configuration of the invention, the at least two magnets, cooperating together and having coaxial magnetic fields, and at least two magnets, cooperating together and having magnetic fields that are not coaxial, are positioned on both opposite sides of the vessel.
In a particular configuration of the invention, the magnets are interdependent with one support.
In another particular configuration of the invention, the magnets intended to the separation and the magnets intended to the mixing are identical.
In one embodiment, the support can be moved in rotation around (to pass from separation to mixing configurations) and longitudinally along (to realize the mixing step) an axle passing through each magnet, the axle being parallel to the moving, defined previously, and perpendicularly to magnet's pole axis.
In a particular configuration of the invention, the magnets intended to the separation and the magnets intended to the mixing are different.
According to any of the former particular configurations of the invention, the lower compartment is constituted by:                one medium compartment to which the magnetic fields are applied successively for washing the magnetic particles, and        one bottom compartment in which the magnetic particles are concentrated and to which the magnetic fields are applied successively to bring the particles into contact with the elution buffer.        
In one particular configuration, in the main process, above disclosed, between step e) and step f), the following intermediate steps are realized:                e1) transferring the separated and mixed magnetic particles from said medium compartment to said bottom compartment,        e2) removing the washing liquid from the vessel, and        e3) adding an elution buffer to the bottom compartment.        
In one particular configuration, in the main process, above disclosed, the following further steps are realized after step f):                g) transferring the magnetic particles from said bottom compartment to the medium compartment or to the upper compartment,        h) removing the elution buffer present in the bottom compartment and containing the entity of interest for further processing.        
In a preferential configuration, the volume of the medium compartment is smaller compared to the upper compartment and bigger compared to the bottom compartment.
In a particular configuration, the magnet brings about the transfer of magnetic particles.
According to any of the former particular configurations of the invention, the molecules of interest are constituted by nucleic acids (RNA and/or DNA).
On the one hand, the binding of the nucleic acids is non-specific and realized directly onto the surface of the magnetic particles.
On the other hand, the binding of the nucleic acids is specific and realized directly onto capture probes carried by the surface of the magnetic particles.
The present invention also relates to a device for extracting possible molecules of interest from a fluid to which is added a suspension of magnetic or (super) paramagnetic particles contained in at least one reaction vessel and capable to bind the molecules of interest comprising                at least one separating station to capture the magnetic particles present in a large upper compartment of each reaction vessel,        at least one washing station to mix said magnetic particles present in a medium compartment of each reaction vessel,        at least one concentrating station to mix said magnetic particles present in a bottom compartment of each reaction vessel, and        at least one pipetting means to dispense and/or to remove part or all of the fluid, the washing liquid and output buffer that are needed to support the process as above presented.        
In one embodiment of the device, the separating station comprises at least two magnets, the pole axis of the magnets forming together an angle different from 180°, preferably included between 60 and 150° and more preferably included between 80 and 120°.
In another embodiment of the device, the washing station comprises at least two magnets, the pole axis of the magnets being parallel one to the other.
The invention also relates to a reaction vessel that can be used in an extracting device, above exposed, comprising:                a top aperture,        one upper compartment with a funnel shape,        one medium compartment with a substantially constant cross-section,        one bottom compartment with a substantially constant cross-section,        a closed base, and        a longitudinal axis defined by the lower compartment.        
According to one embodiment of the reaction vessel, each of the opposite walls of the upper compartment, constituting the funnel shape, is perpendicular to the pole axis of the magnets that is present with respect to this wall.
According to another embodiment of the reaction vessel, the opposite walls of the upper compartment form together an angle different from 180°, preferably included between 60 and 150° and more preferably included between 80 and 120°.
According to any of the embodiment of the reaction vessel above described, the volume of the medium compartment is smaller compared to the upper compartment (and bigger compared to the bottom compartment.
Again according to any of the embodiment of the reaction vessel above described, the ratio between the volumes of the medium compartment and the upper compartment or between the volumes of the bottom compartment and the medium compartment is comprised between 1:2 to 1:100, preferably between 1:5 to 1:20, and more preferably 1:10.
The invention also relates to a set of reaction vessels constituted by at least two vessels, preferentially at least five vessels and more preferentially eight vessels, according to any of vessels presented above, said vessels being arranged symmetrically along one line.
According to one embodiment of the set of reaction vessels, it cooperates with at least two tips, preferentially at least five tips and more preferentially eight tips, said tips constituting:                a first pipetting mean to dispense and/or to remove part or all of the fluid,        a second pipetting mean to dispense and/or to remove part or all of the wash liquid, and        a third pipetting mean to dispense and/or to remove part or all of output buffer.        
According to one embodiment of the set, the free ends of the tips constituting the first or the second or the third pipetting mean being arranged symmetrically along one line or two lines, the two lines of each arrangement being parallel one to the other.
According to another embodiment of the set, the free ends of:                the tips constituting the first pipetting mean being arranged symmetrically along one line,        the tips constituting the second pipetting mean being arranged symmetrically along one or two lines, and        the tips constituting the third pipetting mean being arranged symmetrically along two lines.        
According to one embodiment of the set, the first pipetting mean, the second pipetting mean and the third pipetting mean constitute one unique, two or even three different pipetting means.
With “mixing” in this context is meant that the particles and the fluid are brought in close contact. The word “mixing” thus means contacting in a very efficient manner, such as when particles would be washed or reacted with components present in the fluid. Mixing, in this context, does not necessarily provide a homogeneous mixture after the process is finished. The particles may, when the magnets are removed, segregate to the bottom of the container in which they are comprised or may be held to the wall of the container in a particular location by the magnets. The mixing process can for example be used to wash the particles or to react the particles with a component of the liquid, or to bind a component of the liquid to a reagent coated on the particles. Likewise, the mixing process may result in the elution of a certain component originally present on the particles into the surrounding liquid. The method of the invention is applicable in each of these processes and provides an efficient, rapid and convenient way of contacting magnetic or magnetizable particles with a volume of a certain fluid.